Recently, tick-borne diseases caused by infection with two new human ehrlichiosis agents: Ehrlichia chaffeensis and human granulocytic ehrlichiosis (HGE) agent are increasingly recognized in the U.S. Ehrlichiae are small obligate intracellular gram-negative bacteria, which infect monocytes/macrophages or granulocytes. The overall goal of the proposed study is to unravel ehrlichiacidal mechanisms in host cells. Hypothesis 1 is that IFN-gamma inhibits monocytic ehrlichiae, but not the HGE agent by iron starvation through down regulation of TfR mRNA, and inhibition of Tf accumulation in ehrlichial inclusions. Hypothesis 2 is that ehrlichiae are inhibited by intracellular Ca2+-calmodulin modulating agents or protein kinase inhibitors, because establishment of ehrlichial replication-competent inclusion and/or its metabolism including iron acquisition, is Ca2+ and calmodulin and tyrosine phosphorylation dependent. Hypothesis 3 is that protein kinase A or adenylate cyclase inhibitors potentiate ehrlichiacidal effects, because these inhibitors abrogate the inactivation by ehrlichiae of the host cell's innate immunity including Jak-Stat pathway and reactive oxygen intermediate (ROI) generation. To test these three hypotheses, the Principal Investigator proposes the following aims: Aim1. To examine the influence of IFN-gamma on transfer of 59FE from holoTf to Ehrlichia sp., on TfR mRNA expression and TfR accumulation in ehrlichial inclusions, and on the activity of cytoplasmic iron regulatory factors. Aim 2. To evaluate the influence of Ca2+ ionophore, inhibitors of intracellular Ca2+ mobilization, Ca2+ channel blockers, calmodulin antagonists, phospholipase C inhibitor, and protein tyrosine kinase inhibitors on Ca2+ and inositol phosphates concentrations, localization of proteins involved in membrane docking, trafficking, and fusion, TfR and lysosome localization, and the proteins transglutaminated in ehrlichia-infected cells. Aim 3. To examine mechanisms and roles of protein kinase activation A activation in ehrlichial infection by measuring cAMP levels, adenylate cyclase, phosphodiesterase, and protein kinase A activities under various conditions, and evaluating the influence of protein kinase A inhibitors on IFN-gamma ,A23187-, calmodulin antagonists-, or ROI-induced ehrlichial killing. This study will generate important knowledge on how ehrlichiae usurp the host cell's signaling and membrane sorting mechanisms for their survival. New data on the HGE agent and insights towards interference of the host cell's activation signal as a mechanism of intracellular survival will be gained. The knowledge will be exploited in identifying novel therapeutic and vaccine targets.